S105: CLONAL DIFFERENTIATION TRAJECTORIES OF ADULT ACUTE LYMPHOBLASTIC LEUKEMIA AT SINGLE CELL LEVEL
Authors: Alec Geßner, Adrien Jolly, Tjeerd Sijmonsma, Weijia Yu, Khalil Abou-El-Ardat, Manuel Kaulich, Stefan Guenther, Fabian Lang, Florian Buettner, Michael Rieger
Journal: HemaSphere (2023)

Topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research Background: While remission rates are >90% after first-line therapy in adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL), the prognosis after relapse is dismal with survival rates under 10%. Relapse may arise from remaining therapy-resistant leukemia-initiating cells (LICs) exhibiting specific gene expression pattern. Aims: Consequently, the identification of the cellular architecture and phenotypical plasticity in BCP-ALL in vivo is of key importance in the development of successful treatment strategies. Methods: To investigate subclonal architecture, clonal interference and differentiation trajectories in BCP-ALL, we developed complex genetic barcoding systems at genomic and transcriptomic level to track individual LICs and their progeny. Thereby, we can detect the single-cell barcode together with cellular mRNAs and >50 surface markers using single-cell CITE-Seq. We transduced established patient-derived primary B-ALLs (PDLTCs) with lentiviral barcoding constructs and analyzed leukemic cells before transplantation and after in vivo leukemogenesis in NSG mice at different organ locations. While the majority of freshly transduced PDLTCs homed and engrafted in murine bone marrow and spleen seven days after transplantation, only a few hundred clones seemed to remain at full-blown leukemia. Dominant subclones demonstrated a strong local expansion accounting for most of the tumor mass, whereas hundreds of small clones persisted throughout the disease without expansion. Results: Single-cell RNA-Seq revealed that several weeks after transduction with the barcoding construct, the transcriptomes of cells carrying an identical barcode were highly correlated. This demonstrated that self-renewal and clonal memory in LICs is conserved across multiple generations. Furthermore, the single cell molecular profile of barcoded cells isolated from the bone marrow at full-blown leukemia showed that specific clones undergo differentiation in BCP-ALL and thereby multiple cell surface and gene expression phenotypes can be observed. After transplanting the same pool of stably barcoded cells into several mice, numerous barcodes were detected after leukemogenesis in more than one mouse; strikingly the cells harboring the same barcode were found to exhibit similar differentiation patterns across animals. This result indicates that clones have intrinsic differentiation properties. To validate differentiation states derived from scCITE-Seq, we transplanted prospectively isolated subpopulations into NSGs. Thereby we demonstrated that particular phenotypes are associated with delayed initiation and differential organ engraftment of the disease. Summary/Conclusion: Overall, our work suggests a cellular hierarchy in BCP-ALL with a limited number of LICs. These results have consequences on our understanding of the pathology of the disease and on future treatment strategies. To investigate the cell intrinsic capabilities versus the influence of the environment on the establishment of these phenotypes we will perform whole bone marrow imaging and spatial transcriptomics to determine the localization of these subpopulations and their association with the different niche components. Keywords: Bone Marrow, Clonality, B cell acute lymphoblastic leukemia